Trinucleotide repeat expansions encoding polyglutamine the recently been identified as a novel mechanism of inherited neurological disease. The mechanisms by which polyglutamine expansions lead to neuronal cell death are unclear. Machado-Joseph disease is a progressive neurodegenerative disorder found to result from CAG trinucleotide repeat expansion within the MJD1 gene on human chromosome 14q32.1. The function of the native protein encoded by the MJD gene is unknown, and it bears no resemblance to other characterized proteins in sequence databases. In this proposal, experiments are presented to investigate the mechanisms of MJD polyglutamine neurotoxicity. These studies are anticipated to have broader implications for understanding neurodegenerative disorders, in general, and polyglutamine diseases as a class of novel disorders. Antibodies recognizing the MJD protein and cloned MJD cDNAs will be used as tools for the investigation of MJD protein function and mechanisms of pathogenesis. First, a neuroanatomical characterization of the distribution of MJD mRNA and protein expression will be performed. Tissue specific westerns and northerns will be performed to delineate MJD expression in mammalian body tissues. Second, possible relationships between NMDA excitotoxicity and polyglutamine disorder pathogenesis will be examined. Studies will be performed to determine whether MJD expression correlates with that of neuronal nitric oxide synthase, a defined mediator of NMDA excitotoxicity. Colocalization studies for MJD and nNOS will be pursued with in situ hybridization and immunohistochemistry. Coordinated modulation of MJD expression and nNOS induction will be measured following NMDA treatments of primary neuronal cultures. The results of these studies will be compared to replicate experiments in brain and cultured neurons derived from the nNOS knockout mouse. Lastly, polyglutamine-induced neurotoxicity will be addressed by adenoviral mediated delivery of expanded MJD transgenes to neuronal cultures. Cell death pathways will be monitored. Once appropriate candidate block points are identified, interventions will be performed to attenuate polyglutamine-induced apoptosis.